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Research ArticleDrug Discovery and Translational Medicine

Differential Impact of Amino Acid Substitutions on Critical Residues of the Human Glucagon-Like Peptide-1 Receptor Involved in Peptide Activity and Small-Molecule Allostery

Cassandra Koole, Denise Wootten, John Simms, Laurence J. Miller, Arthur Christopoulos and Patrick M. Sexton
Journal of Pharmacology and Experimental Therapeutics April 2015, 353 (1) 52-63; DOI: https://doi.org/10.1124/jpet.114.220913
Cassandra Koole
Drug Discovery Biology Laboratory, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (C.K., D.W., J.S., A.C., P.M.S.); and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (L.J.M.)
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Denise Wootten
Drug Discovery Biology Laboratory, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (C.K., D.W., J.S., A.C., P.M.S.); and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (L.J.M.)
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John Simms
Drug Discovery Biology Laboratory, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (C.K., D.W., J.S., A.C., P.M.S.); and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (L.J.M.)
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Laurence J. Miller
Drug Discovery Biology Laboratory, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (C.K., D.W., J.S., A.C., P.M.S.); and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (L.J.M.)
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Arthur Christopoulos
Drug Discovery Biology Laboratory, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (C.K., D.W., J.S., A.C., P.M.S.); and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (L.J.M.)
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Patrick M. Sexton
Drug Discovery Biology Laboratory, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (C.K., D.W., J.S., A.C., P.M.S.); and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (L.J.M.)
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Abstract

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein–coupled receptor that has a critical role in the regulation of glucose homeostasis, principally through the regulation of insulin secretion. The receptor system is highly complex, able to be activated by both endogenous [GLP-1(1–36)NH2, GLP-1(1–37), GLP-1(7–36)NH2, GLP-1(7–37), oxyntomodulin], and exogenous (exendin-4) peptides in addition to small-molecule allosteric agonists (compound 2 [6,7-dichloro-2-methylsulfonyl-3-tert-butylaminoquinoxaline], BETP [4-(3-benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine]). Furthermore, the GLP-1R is subject to single-nucleotide polymorphic variance, resulting in amino acid changes in the receptor protein. In this study, we investigated two polymorphic variants previously reported to impact peptide-mediated receptor activity (M149) and small-molecule allostery (C333). These residues were mutated to a series of alternate amino acids, and their functionality was monitored across physiologically significant signaling pathways, including cAMP, extracellular signal-regulated kinase 1 and 2 phosphorylation, and intracellular Ca2+ mobilization, in addition to peptide binding and cell-surface expression. We observed that residue 149 is highly sensitive to mutation, with almost all peptide responses significantly attenuated at mutated receptors. However, most reductions in activity were able to be restored by the small-molecule allosteric agonist compound 2. Conversely, mutation of residue 333 had little impact on peptide-mediated receptor activation, but this activity could not be modulated by compound 2 to the same extent as that observed at the wild-type receptor. These results provide insight into the importance of residues 149 and 333 in peptide function and highlight the complexities of allosteric modulation within this receptor system.

Footnotes

    • Received October 20, 2014.
    • Accepted January 26, 2015.
  • ↵1 Current affiliation: Department of Pharmacology, Aston University, Birmingham, United Kingdom.

  • This work was funded by National Health and Medical Research Council of Australia (NHMRC) project [Grant 1061044, 1065410] and program [Grant 1055134] grants. P.M.S. and A.C. are Principal Research Fellows of the NHMRC.

  • dx.doi.org/10.1124/jpet.114.220913.

  • ↵Embedded ImageThis article has supplemental material available at jpet.aspetjournals.org.

  • Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 353 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 353, Issue 1
1 Apr 2015
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Research ArticleDrug Discovery and Translational Medicine

GLP-1R Residues Important for Activity and Modulation

Cassandra Koole, Denise Wootten, John Simms, Laurence J. Miller, Arthur Christopoulos and Patrick M. Sexton
Journal of Pharmacology and Experimental Therapeutics April 1, 2015, 353 (1) 52-63; DOI: https://doi.org/10.1124/jpet.114.220913

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Research ArticleDrug Discovery and Translational Medicine

GLP-1R Residues Important for Activity and Modulation

Cassandra Koole, Denise Wootten, John Simms, Laurence J. Miller, Arthur Christopoulos and Patrick M. Sexton
Journal of Pharmacology and Experimental Therapeutics April 1, 2015, 353 (1) 52-63; DOI: https://doi.org/10.1124/jpet.114.220913
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